Purification and Properties of L-Methionine γ-Lyase from Aeromonassp.

Four types of β-xylosidases from a concentrated culture filtrate of Pénicillium wortmanni IFO 7237, designated as xylosidase-1, -2, -3, and -4 were purified to homogeneity on SDS polyacrylamide gel electrophoresis by an alcohol precipitation, DEAE-Sephadex A-25 ion exchange chromatography, and isoelectric focusing. The molecular weights of xylosidase-1, -2, -3, and -4 were estimated to be 110,000, 195,000, 210,000, and 180,000 respectively and their isoelectric points to be 3.7, 4.28, 4.6, and 4.8. The pH optima of β-xylosidase activities were from 3 to 4.5. The optimum temperature for enzyme activities was from 55°C to 65°C. On the enzymic hydrolysis of phenyl ß-d- xyloside, the reaction product of each enzyme was found to be β-d-xylose with retention of configuration. All the four ß-xylosidases were free of α-xylosidase and ß-glucosidase activities. All the enzyme activities of four β-xylosidases were strongly inhibited by Hg²⁺ and N- bromosuccinimide. With respect to the hydrolysis patterns and HPLC analysis of hydrolyzates from xylooligosaccharides, xylosidase-2 was totally different from other three as a distinct enzyme. Xylosidase-1 was also in a separate group although xylosidase-3 and -4 showed closely related action patterns as a different group.     

Enzymatic Synthesis of s-Substituted l-Cysteines with Tryptophan Synthase of Escherichia coli

The α₂β₂ complex of tryptophan synthase from Escherichia coli catalyzes β-replacement reactions of l-serine and its derivatives (e.g., β-chloro-l-alanine and O-methyl-Dl-serine) with various alkanethiols. The products from thiobenzyl alcohol and ethanethiol were isolated to demonstrate the enzymatic synthesis of the corresponding S-substituted l-cysteines. Reactivities of various S-substituent donors were examined, and thiols such as thiobenzyl alcohol, 1-propanethiol and 1-butanethiol were found to be much more efficient substituent donors than the physiological substrate, indole. In addition, tryptophan synthase catalyzes β-replacement reactions of l-threonine with thiols to form the corresponding S-substituted β-methylcysteines, which are also produced by β-addition reactions of l-vinylglycine with thiols. These enzymatic reactions facilitate the synthesis of various sulfur-containing amino acids.     

Action of S-Carbamoyl and S-Thiocarbamoyl Derivatives of L-Cysteine on L-Amino Acid Oxidase

Methods are investigated for evaluating the kinetic parameters in a modified Monod’s equation which give the best fit to the growth thermograms for bacterial cultures observed in batch calorimeters. Four mathematical methods were employed as parameter fitting techniques. The growth thermograms observed for soil microbes cultured with glucose as a limiting substrate were used as the objects of the analysis. For the calculation of the heat evolution rate, the Runge-Kutta method, which is commonly used for the numerical analysis, was employed. A comparison of the results obtained by the four methods in terms of closeness of fit to the actual thermograms showed that optimization by direct searching with the Simplex method is the most effective procedure for obtaining the best values of the parameters to reproduce the observed thermograms.     

Flavonoids in Poncirus Hybrids

α_sl-Casein can be made either soluble or insoluble by adjusting the concentration of coexisting calcium ions. In this study, we tried to make a soluble-insoluble interconvertible enzyme through the formation of a conjugate of an enzyme and α_sl-casein using a heterobifunctional crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The conjugate of phosphoglyceromutase and native α_s1-casein did not exhibit sufficient calcium-dependent precipitation. However, conjugates of enzymes (phosphoglyceromutase, enolase or peroxidase) and α_sl-casein polymerized by transglutaminase precipitated almost completely in the presence of more than 50 mM CaCl₂. Most of the enzyme conjugates precipitated as calcium caseinates could be solubilized reversibly with EDTA, without a significant loss of activity. A mixture of the enzyme ? polymerized α_s1-casein conjugates prepared with phosphoglyceromutase, enolase and pyruvate kinase could catalyze sequential reactions which convert d-3-phosphoglycerate into pyruvate with the same efficiency as a mixture of free enzymes. These results indicate that conjugates of enzymes and polymerized α_s1-casein can be useful as soluble-insoluble interconvertible enzymes.     

Leucine Dehydrogenase of a Thermophilic Anaerobe,Clostridium thermoaceticum: Gene Cloning,Purification and Characterization

The leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of 46%. The enzyme of E. coli- pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 15 min; at 55°C and various pH’s between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD⁺ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotin- amide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].     

Isolation and Characterization of Globulin from Seeds of Amaranthus hypochondriacus L.

Seeds of grain amaranths contain a high amount (about 60% of total nitrogen) of albumin and globulin and a trace amount of prolamin. From salt-soluble extracts of A. hypochondriacus seeds, a globulin (440,000 in apparent molecular weight and ) was purified by Sepharose 6B gel and DEAE-cellulose column chromatographies. The protein comprised at least four kinds of subunits whose molecular weights were 36,000, 32,000, 20,000 and 18,000, respectively. The amino acid composition of the globulin was almost similar to those of soybean and oat globulins.     

Structure and Bitterness in Flavanone Glycosides

Naringenin-7-β-kojibioside, -7-β-sophoroside, -7-[α-d-galactosyl(l→2)β-d-glucoside], -7-[β-d-glucosyl(l→2)β-d-galactoside], and also hesperetin-7-β-kojibioside and -7-β-sophoroside were prepared by the coupling of naringenin or hesperetin with the α-acetobromo derivatives of the appropriate disaccharides, followed by saponification.

Their relative bitterness values were discussed in comparison with naringin and neo-hesperidin.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Flavonoids in Citrus and Related Genera

The occurence and distribution of flavonoid glycosides in young leaves and young and mature fruits of many citrus species and trifoliate orange were investigated. The occurence of neohesperidin in both young leaves and young fruits is fairly common to a number of species in subgenus Archicitrus. Ripe fruits of citrus could be classified into (a) the hesperidin group (b) the neohesperidin group (c) the naringin group and (d) the isonaringin group. A new flavanone glycoside, isonaringin, isolated from young fruits of Jagatarayu and Teng mikan is slightly bitter and has been determined by chemical and spectral evidences to have the structure of naringenin-7-rhamnoglucoside. Data showing the occurence of flavanone glycosides in some artificial citrus hybrids were also given.     

Novel mutations of CDKN1C in Japanese patients with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease that is characterized by macrosomia, macroglossia, abdominal wall defects, and variable minor features. BWS is caused by several genetic/epigenetic alterations, such as loss of methylation at KvDMR1, gain of methylation at H19-DMR, paternal uniparental disomy of chromosome 11, CDKN1C mutations, and structural abnormalities of chromosome 11. CDKN1C is an imprinted gene with maternal preferential expression, encoding for a cyclin-dependent kinase (CDK) inhibitor. Mutations in CDKN1C are found in 40 % of familial BWS cases with dominant maternal transmission and in ~5 % of sporadic cases. In this study, we searched for CDKN1C mutations in 37 BWS cases that had no evidence for other alterations. We found five mutations—four novel and one known—from a total of six patients. Four were maternally inherited and one was a de novo mutation. Two frame-shift mutations and one nonsense mutation abolished the QT domain, containing a PCNA-binding domain and a nuclear localization signal. Two missense mutations occurred in the CDK inhibitory domain, diminishing its inhibitory function. The above-mentioned mutations were predicted by in silico analysis to lead to loss of function; therefore, we strongly suspect that such anomalies are causative in the etiology of BWS.